Cloning Of Rolled Leaf Gene Rl_（t） And The RNAi Analysis Of OsAGO1a In Rice

Leafs play important roles in the plant development for their function of photosynthesis. Leaf character (length, width, leaf rolling index and so on) is one of the main concerns in high yield breeding in rice, which affects the plant morphology and is related to the shaping of plant type. The direct effect of rolled leaf is straightening the plant leaf, thus, optimizing the canopy light transmission, improving condition of sunlight reception in the middle and lower parts of population of rice plants at middle and late stages of growth, and increasing the yield in rice. However, excessive rolled leaf will greatly weaken the light-received intensity per unit area, which is disadvantageous to the photosynthesis of crown canopy. In this study, we cloned the rolled leaf gene rl(t), and researched the moderate leaf rolling index, on the basic of previous studies. Moreover, we identified a new rolled leaf gene OsAGO1a by reverse genetics method. Finally, we found a novel method of the reconstruction of the multiple clonal sites (MCS) of vector in the process of the functional research. The main results were as follows:1) Moderate leaf rolling is one of the most important morphological traits in rice breeding for plant ideotype. Previous studies have shown that the rl(t) gene has higher breeding potential for developing hybrid-rice variety with ideotype, because it leads to appropriate leaf rolling index (LRI) of about30%in heterozygous status and showed positive effect on grain yield. In this study, we isolated rl(t) and preliminary investigated its function in regulation of rice leaf rolling. Through sequencing, we found that only one base variation (G to U) in the fine mapping region (11kb) of rl(t), which located in3’-untranslated region (3’-UTR) of the only predicted gene, Roc5(Rice outermost cell-specific). Expression level of rl(t) is significantly higher in mutant than in wild type. Through RNAi and overexpression analysis, we found that the expression level of rl(t) correlated with LRI and leaf bulliform area, and associated with leaf abaxial or adaxial rolling. These results confirmed that rl(t) controls leaf rolling in a dosage-dependent manner. Bioinformatics analysis revealed a conserved17-nt sequence (called GU-rich element) in3’-UTR of HD-ZIP IV (homeodomain leucine zipper class IV) family genes including rl(t). Based on the model of this element in regulation of mRNA stability in mammals, we speculate that the base substitution in this element accounts for the higher expression of rl(t) in mutant than that in wild type, and ultimately leads to adaxial rolling of leaf. The discoveries provide a new insight for elucidating the molecular mechanism of leaf rolling controlled by rl(t).2) Eight rice NILs with different leaf rolling index (LRI) under the background of Nipponbare have been obtained by regulating expression of dosage-dependent rolled leaf gene ri(t). These NILs were used to analyze the relationship between LRI and economic traits. The result of the model of LRI and yield showed that, the yield of Nipponbare was quadratically correlated with the LRI, and the yield reached the peak when the LRI was about5.9%. These results suggested that, the most appropriate LRI was about5.9%. The feasibility of strategy has been studied and discussed to define the most appropriate LRI of different rice varieties.3) Argonaute (AGO) protein is the core effectors in RNA-induced silencing complexes (RISCs), playing an important role in the development of plant. The2th and the11th-12th exon fragments of OsAGO1a were amplified independently to make two RNAi constructs: OsAGO1aI-1E and OsAGO1aI-2E. To clarify the function of OsAGO1a in the development of rice, the expression of OsAGO1a has been suppressed by introducing RNAi constructs to Nipponbare through Agrobacterium-mediated transformation. The result showed that reduced expression of OsAGO1a lead to adaxial leaf rolling without affecting main agronomic traits.4) In order to reconstruct the vector MCS easily, the DNA fragment was amplified with primer pairs, whose5’ends with several restriction sites. Then the DNA fragment was inserted into MCS of vector after digestion with endonuclease in its both sides. This method was named as adaptor primer-PCR introducing. Digestion with endonuclease proves that the designed restriction sites have been inserted into MCS of the vector and the restriction sites were digestible. These results showed that the adaptor primer-PCR introducing method was a feasible technique to reconstruct the vector MCS.